Gene-X-ppt(Gene10-基因十)--Chapter26

Chapter26TheOperon26.1Introduction•coupledtranscription/translation–ThephenomenainbacteriawheretranslationofthemRNAoccurssimultaneouslywithitstranscription.•operon–Aunitofbacterialgeneexpressionandregulation,includingstructuralgenesandcontrolelementsinDNArecognizedbyregulatorgeneproduct(s).26.1Introduction•trans-acting–AproductthatcanfunctiononanycopyofitstargetDNA.ThisimpliesthatitisadiffusibleproteinorRNA.•cis-acting–AsitethataffectstheactivityonlyofsequencesonitsownmoleculeofDNA(orRNA);thispropertyusuallyimpliesthatthesitedoesnotcodeforprotein.26.1Introduction•regulatorgene–Agenethatcodesforaproduct(typicallyprotein)thatcontrolstheexpressionofothergenes(usuallyattheleveloftranscription).•structuralgene–AgenethatcodesforanyRNAorproteinproductotherthanaregulator.FIGURE01:AregulatorbindsatargetsiteonDNA26.1Introduction•Innegativeregulation,arepressorproteinbindstoanoperatortopreventagenefrombeingexpressed.•Inpositiveregulation,atranscriptionfactorisrequiredtobindatthepromoterinordertoenableRNApolymerasetoinitiatetranscription.FIGURE02:ArepressorstopsRNApolymerasefrominitiatingFIGURE03:TranscriptionfactorsenableRNApolymerasetobindtothepromoter26.1Introduction•Ininducibleregulation,thegeneisregulatedbythepresenceofitssubstrate(theinducer).•Inrepressibleregulation,thegeneisregulatedbytheproductofitsenzymepathway(thecorepressor).26.1Introduction•Wecancombinetheseinallfourcombinations:negativeinducible,negativerepressible,positiveinducible,andpositiverepressible.FIGURE04:Inductionandrepressioncanbeunderpositiveornegativecontrol26.2StructuralGeneClustersAreCoordinatelyControlled•GenescodingforproteinsthatfunctioninthesamepathwaymaybelocatedadjacenttooneanotherandcontrolledasasingleunitthatistranscribedintoapolycistronicmRNA.FIGURE05:Thelacoperonincludescis-actingregulatorelementsandprotein-codingstructuralgenes26.3ThelacOperonIsNegativeInducible•TranscriptionofthelacZYAoperoniscontrolledbyarepressorprotein(thelacrepressor)thatbindstoanoperatorthatoverlapsthepromoteratthestartofthecluster.•constitutiveexpression–Astateinwhichageneisexpressedcontinuously.•Intheabsenceofβ-galactosides,thelacoperonisexpressedonlyataverylow(basal)level.FIGURE06:Thepromoterandoperatoroverlap26.3ThelacOperonIsNegativeInducible•TherepressorproteinisatetramerofidenticalsubunitscodedbythelacIgene.•β-galactosidesugars,thesubstratesofthelacoperon,areitsinducer.•Additionofspecificβ-galactosidesinducestranscriptionofallthreegenesofthelacoperon.•ThelacmRNAisextremelyunstable;asaresult,inductioncanberapidlyreversed.FIGURE07:lacexpressionrespondstoinducer26.4lacRepressorIsControlledbyaSmall-MoleculeInducer•Aninducerfunctionsbyconvertingtherepressorproteinintoaformwithloweroperatoraffinity.•Repressorhastwobindingsites,onefortheoperatorDNAandanotherfortheinducer.•gratuitousinducer–Inducersthatresembleauthenticinducersoftranscription,butarenotsubstratesfortheinducedenzymes.FIGURE08:Arepressortetramerbindstheoperatortopreventtranscription26.4lacRepressorIsControlledbyaSmall-MoleculeInducer•RepressorisinactivatedbyanallostericinteractioninwhichbindingofinduceratitssitechangesthepropertiesoftheDNA-bindingsite(allostericcontrol).•Thetrueinducerisallolactose,nottheactualsubstrateofβ-galactosidase.FIGURE09:Inducerinactivatesrepressor,allowinggeneexpression26.5cis-ActingConstitutiveMutationsIdentifytheOperator•Mutationsintheoperatorcauseconstitutiveexpressionofallthreelacstructuralgenes.•Thesemutationsarecis-actingandaffectonlythosegenesonthecontiguousstretchofDNA.•MutationsinthepromoterpreventexpressionoflacZYAareuninducibleandcis-acting.26.5cis-ActingConstitutiveMutationsIdentifytheOperator•cis-dominant–AsiteormutationthataffectsthepropertiesonlyofitsownmoleculeofDNA,oftenindicatingthatasitedoesnotcodeforadiffusibleproduct.FIGURE10:Constitutiveoperatormutantcannotbindrepressorprotein26.6trans-ActingMutationsIdentifytheRegulatorGene•MutationsinthelacIgenearetrans-actingandaffectexpressionofalllacZYAclustersinthebacterium.•MutationsthateliminatelacIfunctioncauseconstitutiveexpressionandarerecessive(lacI–).•MutationsintheDNA-bindingsiteoftherepressorareconstitutivebecausetherepressorcannotbindtheoperator.FIGURE11:Defectiverepressorcausesconstitutiveexpression26.6trans-ActingMutationsIdentifytheRegulatorGene•Mutationsintheinducer-bindingsiteoftherepressorpreventitfrombeinginactivatedandcauseuninducibility.•Whenmutantandwild-typesubunitsarepresent,asinglelacI–dmutantsubunitcaninactivateatetramerwhoseothersubunitsarewild-type.–Itisdominantnegative.26.6trans-ActingMutationsIdentifytheRegulatorGene•interalleliccomplementation–Thechangeinthepropertiesofaheteromultimericproteinbroughtaboutbytheinteractionofsubunitscodedbytwodifferentmutantalleles.–Themixedproteinmaybemoreorlessactivethantheproteinconsistingofsubunitsofonlyoneortheothertype.26.6trans-ActingMutationsIdentifytheRegulatorGene•negativecomplementation–Thisoccurswheninteralleliccomplementationallowsamutantsubunittosuppresstheactivityofawild-typesubunitinamultimericprotein.•lacI–dmutationsoccurintheDNA-bi